Core-shell nanoparticles with multiple cores and a method for fabricating them

ABSTRACT

The present invention is directed toward core-shell nanoparticles, each comprising a ligand-capped metal shell surrounding a plurality of discrete, nonconcentric, metal-containing cores. Methods of making and using these nanoparticles are also disclosed.

This application is a division of U.S. patent application Ser. No.13/680,749, filed Nov. 19, 2012, now allowed, which is a division ofU.S. patent application Ser. No. 12/034,155, filed Feb. 20, 2008, nowU.S. Pat. No. 8,343,627, issued Jan. 1, 2013, which claims the benefitof U.S. Provisional Patent Application Ser. No. 60/890,699, filed Feb.20, 2007, which are hereby incorporated by reference in their entirety.

This invention was made with government support under grant numberCHE0349040 awarded by the National Science Foundation. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to core-shell nanoparticles with multiplecores and a method for fabricating them.

BACKGROUND OF THE INVENTION

Nanoparticles exhibit intriguing changes in electronic, optical, andmagnetic properties as a result of the nanoscale dimensionality (Danielet al., Chem. Rev. 104:293 (2004); Xia et al., Adv. Mater. 12:693(2000)). The ability to engineer size and monodispersity is essentialfor the exploration of these properties. The preparation of magneticnanoparticles and nanocomposites has attracted both fundamental andpractical interest because of potential applications in areas such asferrofluids, medical imaging, drug targeting and delivery, cancertherapy, separations, and catalysis (Kim et al., J. Magn. Magn. Mater.225:256 (2001); Niemeyer, Angew. Chem. Int. Ed. 40:4128 (2001);Neuberger et al., J. Magn. Magn. Mater. 293:483 (2005); Tartaj et al.,J. Magn. Magn. Mater. 290:28 (2005); Dobson, Drug Dev. Res. 67:55(2006)). However, one of the major obstacles is the lack of flexibilityin surface modification and biocompatibility. Gold coating on magneticparticles provides an effective way to overcome such an obstacle viawell-established surface chemistry to impart magnetic particles with thedesired chemical or bio-medical properties (Daniel et al., Chem. Rev.104:293 (2004); Xia et al., Adv. Mater. 12:693 (2000)). There have beenreports on the synthesis of gold-coated magnetic core-shell particles byvarious methods, e.g. gamma ray, laser ablation, sonochemical method,layer-by-layer electrostatic deposition, chemical reduction, and micellemethods (Kinoshita et al., J. Magn. Magn. Mater. 293:106 (2005); Zhanget al., J. Phys. Chem. B 110:7122 (2006); Caruntu et al., Chem. Mater.17:3398 (2005); Spasova et al., J. Mater. Chem. 15:2095 (2005); Stoevaet al., J. Am. Chem. Soc 127:15362 (2005); Lyon et al., Nano Letters4:719 (2004); Mandal et al., J. Colloid Interface Sci. 286:187 (2005)).Recently reported was the synthesis of monodispersed core-shell Feoxide-Au nanoparticles via coating pre-synthesized iron oxidenanoparticles (5-7 nm sizes) with gold shells (1-2 nm) (Wang et al., J.Phys. Chem. B 109:21593 (2005)). However, many of the magnetic core orshell dimensions have been limited to <15 nm. This limitation poses aserious barrier to magnetic applications where the size tunability,especially in larger sizes (up to ˜100 nm) with sufficientmagnetization, is required.

The present invention is directed to overcoming these deficiencies inthe art.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed toward core-shellnanoparticles, each comprising a ligand-capped metal shell surrounding aplurality of discrete, nonconcentric, metal-containing cores.

Another aspect is directed to a method of producing core-shellnanoparticles, each comprising a ligand-capped metal shell surroundingone or more metal-containing cores. This method includes providingligand-capped metal-containing core material nanoparticles andligand-capped metal shell material nanoparticles. These nanoparticlesare reacted under conditions effective to produce the core-shellnanoparticles comprising a ligand-capped metal shell surrounding one ormore metal-containing cores.

A further aspect of the present invention is directed to a method ofseparating a target molecule from a sample. In accordance with thismethod, core-shell nanoparticles, each comprising a ligand-capped metalshell surrounding a plurality of discrete, nonconcentric,metal-containing cores are provided, with a first binding material boundto the ligand-capped shell. The binding material that specifically bindsto the target molecule is incubated with the sample in a reaction vesselunder conditions effective for the first binding material to bind to thetarget molecule. The reaction vessel is contacted with a magnet underconditions effective to immobilize the nanoparticles in the reactionvessel. The immobilized nanoparticles may be recovered.

Disclosed herein is a novel thermal approach to the fabrication ofcore-shell magnetic nanoparticles with not only high monodispersity butalso size tunability in the 5-100 nm range. The basic idea explores theviability of hetero-interparticle coalescence between gold and magneticnanoparticles under encapsulating environment for creating core-shelltype nanoparticles in which the magnetic core consists of single ormultiple metal cores with a pomegranate-like interior structuredepending on the degree of coalescence (see FIG. 1).

This approach is new and differs from previous methods for synthesizinggold-coated Fe-Oxide particles in two significant ways: First, thepresent method uses a thermal evolution method starting from Fe-Oxidenanoparticles and Au nanoparticles as precursors, whereas the previoussynthesis method uses Fe-Oxide nanoparticles and Au(Ac)₃ molecules asprecursors. Second, the present method produces golden magneticparticles with either single core or multiple cores, whereas theprevious method produces golden magnetic particles with only a singlecore.

This approach is also new in comparison with the homo-interparticlecoalescence demonstrated for evolving the sizes of gold nanoparticles bya thermally-activated evolution (Zhong et al, Chem. Comm. 13:1211(1999); Maye et al., Langmuir 16:490 (2000), which are herebyincorporated by reference in their entirety). In the previous methodinterparticle coalescence of metals or alloys is utilized for evolvingthe sizes of gold or alloy nanoparticles. In the present method, it ishetero-coalescence between Fe-oxide nanoparticles and gold nanoparticlesfor evolving single core Fe-Oxide-Au nanoparticles and multiple-core(Fe-Oxide)_(n)-Au nanoparticles. The competition between growing Au,Fe-oxide-Au, and the pomegranate-like core-shell nanoparticles isdetermined by solution temperature, composition and capping structures.This approach could serve as a simple and effective strategy formonodispersed Fe-oxide-Au nanoparticles of controlled sizes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing illustrating the hetero-interparticlecoalescence of nanoparticles. Component 1A represents ligand-cappedmetal shell material nanoparticles. Component 1B representsligand-capped metal-containing core material nanoparticles. Component Aand B combine to form nanoparticles (i.e. Component C) comprising aligand-capped metal shell surrounding a single metal-containing core.Component C coalesces to form component D which is a nanoparticlecomprising a ligand-capped metal shell surrounding a plurality ofdiscrete, nonconcentric, metal-containing cores.

FIG. 2A shows a schematic drawing of a core-shell nanoparticle withbinding material and antibodies bound to the core-shell nanoparticle.FIG. 2B-C are schematic drawings of methods of concentrating targetanalytes using magnetic nanoparticles.

FIGS. 3A-B are TEM micrographs of gold nanoparticles produced by thermalprocessing (149° C.) of Au_(2nm)-DT nanoparticles. FIG. 3A shows theprecursor nanoparticles 2.0±0.4 nm and FIG. 3B shows the productnanoparticles (6.4±0.4 nm).

FIGS. 4A-B are TEM images of OA/OAM-capped Fe₂O₃ nanoparticles producedby thermal processing (149° C.): before (FIG. 4A, 4.4±0.3 nm) and after(FIG. 4B, 4.5±0.5 nm) thermal processing.

FIGS. 5A-C are TEM images for precursor Fe₂O₃ (FIG. 5A), Au (FIG. 5B),and thermally-evolved Au-coated Fe₂O₃ nanoparticles using 25:1 ratio ofAu to Fe₂O₃ nanoparticles (FIG. 5C).

FIGS. 6A-B are TEM images of the nanoparticles thermally evolved fromthe same nanoparticles precursors but with two different molar ratios ofAu nanoparticles to Fe₂O₃ nanoparticles, (FIG. 6A) Au:Fe₂O₃=25:1, and(FIG. 6B) Au:Fe₂O₃=132:1.

FIGS. 7A-D are TEM images of nanoparticles obtained from the sameprecursor molar ratios (Au:Fe₂O₃=5:1) but using different capping agentson Fe₂O₃: OA-capped Fe₂O₃ (FIG. 7A), OA-capped Fe₂O₃ (FIG. 7B),OAM-capped Fe₂O₃ (FIG. 7C), and OA/OAM-capped Fe₂O₃ in a ˜2× reactionvolume (FIG. 7D).

FIG. 8 is a schematic illustration of the dithiolate-gold based bindingchemistry for the thin film assembly of Fe₂O₃@Au (A), Au (B) and Fe₂O₃(C) nanoparticles on a substrate.

FIGS. 9A-B are SERS spectra for MBA label which are incorporated ontoOA-OAM capped Fe₂O₃@Au nanoparticles (FIG. 9A) and protein-A capped Aunanoparticles (FIG. 9B).

FIGS. 10A-C is a photograph showing magnetic properties of Fe₂O₃@Aunanoparticles dissolved in toluene (FIG. 10A), suspended inethanol-toluene (FIG. 10B), and after applying a magnet to thesuspension of FIG. 10B (FIG. 10C).

FIGS. 11A-B are FTIR spectra of OA/OM-capped core@shell Fe₂O₃@Auparticles before (FIG. 11A) and after (FIG. 11B) ligand exchangereaction with MUA.

FIG. 12 is an illustration of the reactions and product separation ofantibody-labeled Fe₂O₃@Au nanoparticles in two different reactionsystems: (A1-B1): reaction with protein A capped gold nanoparticles, and(A2-B2): reaction with BSA capped gold nanoparticles. In both cases, theSERS Label (L) mercaptobenzoic acid (MBA); Antibody (Ab): anti-rabbitIgG.

FIG. 13 is a UV-Vis spectra monitoring the reaction betweenAu/Protein-A/L and Fe₂O₃@Au/Ab (A1). Inset: the spectra monitoring thereaction between Au/BSA/L and Fe₂O₃@Au/Ab (A2). The spectra wererecorded as a function of time (within 1 hr). The arrows indicate thedirection of the spectral evolution as a function of time.

FIG. 14 is a SERS spectra of the products from the reactions betweenAu/Protein-A/L and Fe₂O₃@Au/Ab and between Au/BSA/L and Fe₂O₃@Au/Ab(A2). A magnet was used to collect the particles, which were thendeposited on a Au substrate. SERS Label (L): MBA. Antibody (Ab):anti-rabbit-IgG.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is directed toward core-shellnanoparticles, each comprising a ligand-capped metal shell surrounding aplurality of discrete, nonconcentric, metal-containing cores. Thecore-shell nanoparticles may be present in a monodispersion withcontrolled diameters ranging from 5 nm to 100 nm.

The metal-containing cores may be magnetic, paramagnetic orsuperparamagnetic. The metal of the metal-containing cores may be iron(e.g., Fe₃O₄ or Fe₂O₃), magnesium, cobalt, or mixtures thereof.

The metal of the metal shell may be gold, silver, platinum, rhodium,palladium, vanadium, titanium, iron, cobalt, magnesium, ruthenium,chromium, molybdenum, tantalum, zirconium, manganese, tin, or mixturesthereof.

The capping ligand may be decanethiolate, oleylamine, oleic acid,acrylates, N,N-trimethyl(undecylmercapto)ammonium (TUA),tetrabutylammonium tetrafluoroborate (TBA), tetramethylammonium bromide(TMA), cetyltrimethylammonium bromide (CTAB), citrates, polymethacrylate, ascorbic acid, DNA, 2-mercaptopropionic acid (MPA),3-mercaptopropionic acid (MPA), 11-mercaptoundecanoic acid (MUA),10-mercaptodecane-1-sulfonic acid, 16-mercaptohexadecanoic acid,diimide, N-(2-mercaptopropionyl)glycine (tiopronin), 2-mercaptoethanol,4-mercapto-1-butanol, dodecyl sulfate, amino acids, homocysteine,homocystine, cysteine, cystine, glutathione, mercaptobenzoic acid (MBA),Protein A, bovine serum albumin (BSA), or anti-rabbit-IgG (Ab).

The core-shell nanoparticles may further comprise a binding materialbound to the ligand-capped shell. The binding material may be proteins,peptides, antibodies, or antigens.

Another aspect is directed to a method of producing core-shellnanoparticles, each comprising a ligand-capped metal shell surroundingone or more metal-containing cores. This method includes providingligand-capped metal-containing core material nanoparticles andligand-capped metal shell material nanoparticles. These nanoparticlesare reacted under conditions effective to produce the core-shellnanoparticles comprising a ligand-capped metal shell surrounding one ormore metal-containing cores.

As shown in FIG. 1, the method of the present invention provides forhetero-interparticle coalescence of ligand-capped metal shell materialnanoparticles, e.g., decanethiolate (DT)-capped Au (Component A) andligand-capped metal-containing core material nanoparticles, e.g., oleicacid or oleylamine (OA/OM)-capped Fe₂O₃ (Component B). Thermal evolutionresults in the formation of nanoparticles comprising a ligand-cappedmetal shell surrounding a single metal-containing core (Component C).These single-core nanoparticles can undergo further thermal evolution toform nanoparticles comprising a ligand-capped metal shell surrounding aplurality of discrete, nonconcentric, metal-containing cores (ComponentD).

The specific materials used to form these ligand-capped metal shellsurrounding one or more metal containing cores and the structure of thenanoparticles themselves are substantially the same as those describedabove.

The reaction conditions may include combining the ligand-cappedmetal-containing core material nanoparticles and the ligand-capped metalshell material nanoparticles in a solvent to form a reaction mixture.The reaction mixture is heated under conditions effective to form thecore-shell nanoparticles comprising a ligand-capped metal shellsurrounding one or more metal-containing cores.

The solvent may include toluene, tetraoctylammonium bromide, and/ordecanethiols and may be heated to a temperature of 140-160° C.

The core-shell nanoparticles may be subjected to one or more sizingoperations, such as centrifugation.

A further aspect of the present invention is directed to a method ofseparating a target molecule from a sample. In accordance with thismethod, core-shell nanoparticles, each comprising a ligand-capped metalshell surrounding a plurality of discrete, nonconcentric,metal-containing cores are provided, with a first binding material boundto the ligand-capped shell. The binding material that specifically bindsto the target molecule is incubated with the sample in a reaction vesselunder conditions effective for the first binding material to bind to thetarget molecule. The reaction vessel is contacted with a magnet underconditions effective to immobilize the nanoparticles in the reactionvessel. The immobilized nanoparticles may be recovered.

The method may include removing liquids from the reaction vessel. Oncethe nanoparticles with binding materials are bound to the target in asample solution and the vessel is contacted with a magnet therebyimmobilizing the nanoparticles-target complex, all or some of the samplesolution can be removed for further purification, analysis, or otheruse. The liquids can be removed by various means well known in the artincluding pumping, pouring, pipetting, or evaporation.

Furthermore, the target molecules may be separated from thenanoparticles. Target molecules may be reversibly bound to the bindingmaterial or the binding material may be reversibly bound to thenanoparticles, allowing separation of the target molecules from thenanoparticles by various methods known in the art such as solvation,exchange, heating, or digestion.

The binding material may be proteins, peptides, antibodies, antigens orother suitable material known in the art.

Magnetic separation techniques are commonly used for the purification,quantification, or identification of various substances (see Ito et al.,J. Biosci. Bioeng. 100(1):1-11(2005); Alexiou et al., J. Nanosci.Nanotechnol., 6:2762 (2006); and Risoen et al., Protein Expr. Purif.6(3):272-7 (1995), which are hereby incorporated by reference in theirentirety). The term “magnetic particles” is meant to include particlesthat are magnetic, paramagnetic, or superparamagnetic properties. Thus,the magnetic particles are magnetically displaceable but are notnecessarily permanently magnetizable. Methods for the determination ofanalytes using magnetic particles are described, for example, in U.S.Pat. No. 4,554,088, which is hereby incorporated by reference in itsentirety.

The magnetic particle may be bound to an affinity ligand, the nature ofwhich will be selected based on its affinity for a particular analytewhose presence or absence in a sample is to be ascertained. The affinitymolecule may, therefore, comprise any molecule capable of being linkedto a magnetic particle which is also capable of specific recognition ofa particular analyte. Affinity ligands, therefore, include monoclonalantibodies, polyclonal antibodies, antibody fragments, nucleic acids,oligonucleotides, proteins, oligopeptides, polysaccharides, sugars,peptides, peptide encoding nucleic acid molecules, antigens, drugs, andother ligands.

The target material may optionally be a material of biological orsynthetic origin. For examples, such target materials may be antibodies,amino acids, proteins, peptides, polypeptides, enzymes, enzymesubstrates, hormones, lymphokines, metabolites, antigens, haptens,lectins, avidin, streptavidin, toxins, poisons, environmentalpollutants, carbohydrates, oligosaccharides, polysaccharides,glycoproteins, glycolipids, nucleotides, oligonucleotides, nucleic acidsand derivatised nucleic acids, DNA, RNA, natural or synthetic drugs,receptors, virus particles, bacterial particles, virus components,cells, cellular components, and natural or synthetic lipid vesicles.

Magnetic particles have potential to be used in imaging and analyticaldetection assays as well. For example, in a Surfaced Enhanced RamanSpectroscopy (SERS)-based assay, increasing analyte concentration insolution or local analyte concentration at an assay surface cansignificantly improve the limits of detection of different analytes,especially of large biomolecules such as bacteria and viruses. FIG. 2Ashows a representation of antibodies, for example, bound to a magneticcore-shell nanoparticle. Such a system could be used to bind a targetforming a nanoparticle-target complex. Application of a magnetic fieldwill allow immobilization of the nanoparticle-target complex (FIG. 2B).Alternatively, through the application of a magnetic field, thenanoparticle-target complex can be concentrated at the site of an assaysurface (FIG. 2C) allowing for detection or improvement of the limits ofdetection.

During recent years, there has been in increase in interest regardingthe use of magnetic nanoparticles as contrast agents for use inconjunction with magnetic resonance imaging (MRI) techniques (Ito etal., J. Biosci. Bioeng. 100:1-11 (2005), which is hereby incorporated byreference in its entirety). Magnetic nanoparticles have also beenproposed for use in direct sensing methods for diagnosis of cancer(Suzuki et al., Brain Tumor Pathol. 13:127 (1996), which is herebyincorporated by reference in its entirety) and for novel tissueengineering methodologies utilizing magnetic force and functionalizedmagnetic nanoparticles to manipulate cells (Ito et al., J. Biosci.Bioeng. 100:1-11 (2005), which is hereby incorporated by reference inits entirety).

In therapeutic applications, for example, application of a magneticfield to the patient may serve to target drug-carrying magneticparticles to a desired body site. In many cases, the dose ofsystemically administered chemotherapeutics is limited by the toxicityand negative side effects of the drug. Therapeutically sufficientconcentrations of the drugs in the respective tissues often need to bequite high. Magnetic carrier systems should allow targeted drug deliveryto achieve such high local concentrations in the targeted tissues,thereby minimizing the general distribution throughout the body. Specialmagnetic guidance systems can direct, accumulate, and hold the particlesin the targeted area, for example, a tumor region (Alexiou et al., J.Nanosci. Nanotechnol. 6:2762 (2006), which is hereby incorporated byreference in its entirety).

EXAMPLES

The following examples are intended to illustrate the invention, and arenot intended to limit its scope.

Example 1 Chemicals

Iron pentacarbonyl (Fe(CO)₅), phenyl ether, trimethylamine oxide,decanethiol (DT) tetraoctylammonium bromide (TOA-Br), oleylamine (OAM),oleic acid (OA), trimethylamine oxide dihydreate ((CH₃)₃NO.2H₂O), bovineserum albumin (BSA), 11-mercaptoundecanoic acid (MUA), mercaptobenzoicacid (MBA), dithiobis (succinimidyl propionate) (DSP), and othersolvents (hexane, toluene, and ethanol) were obtained form Aldrich andwere used as received. Anti-rabbit IgG and rabbit IgG were purchasedfrom Pierce. Protein A and gold nanoparticles were obtained from TedPella.

Example 2 Synthesis and Preparation

Fe₂O₃ (γ-Fe₂O₃) nanoparticles and Au nanoparticles were synthesized byknown protocols, whereas the preparation of Fe₂O₃@Au nanoparticles wasbased on a new protocol developed in this work. For the synthesis ofFe₂O₃ nanoparticles, Fe₂O₃ nanoparticles capped with OA (and/or OAM)were prepared based on the modified procedure reported previously (Wanget al., J Phys. Chem. B 109:21593 (2005), which is hereby incorporatedby reference in its entirety). Briefly, 0.74 mL of Fe(CO)₅ and 5.3 mL ofOA (and/or OAM) in 40 mL of phenyl ether was stirred at 100° C. underargon purge. The solution was heated to 253° C. and refluxed for 1 h.The solution turned to dark brown. After the solution was cooled to roomtemperature, 1.26 g of (CH₃)₃NO.2H₂O was added and stirred at 130° C.for 2 h. Temperature was increased to 253° C. and refluxed for 2 h. Thereaction solution was stirred overnight. The resulting nanoparticleswere precipitated with ethanol and rinsed multiple times. Finally,particles were dispersed in hexane or toluene. For the synthesis of Aunanoparticles, the standard two-phase method reported by Brust andSchriffrin (J. Chem. Soc., Chem. Commun. 1994:801-802, which is herebyincorporated by reference in its entirety) was used. Gold nanoparticlesof 2 nm diameter encapsulated with DT monolayer shells (Au_(2nm)-DT)were synthesized.

For the preparation of Fe₂O₃@Au nanoparticles, a modified strategy ofthermally-activated processing protocol (Schadt et al., Chem. Mater.18:5147 (2006); Maye et al., Langmuir 16:490 (2000); Zhong et al., Chem.Commun. 13:1211 (1999), which are hereby incorporated by reference intheir entirety) was used. The thermal processing treatment of Aunanoparticles involved molecular desorption, nanoscrystal corecoalescence, and molecular re-encapsulation processes in the evolutionof nanoparticle precursors at elevated temperatures (149° C.). Thethermal processing of small-sized monolayer-protected nanoparticles asprecursors (Schadt et al., Chem. Mater. 18:5147 (2006); Maye et al.,Langmuir 16:490 (2000); Zhong et al., Chem. Commun. 13:1211 (1999),which are hereby incorporated by reference in their entirety) hasrecently gained increasing interest for processing nanoparticle size andmonodispersity (Clarke et al., Langmuir 17:6048-6050 (2001); Teranishiet al., Adv. Mater. 13:1699-1701 (2001); Fan et al., Chem. Commun.2006:2323-2325; Terzi et al., J. Phys. Chem. B 109:19397-19402 (2005);Shaffer et al., Langmuir 20:8343-8351 (2004); Chen et al., J. Phys.Chem. B 105:8816-8820 (2001), which are hereby incorporated by referencein their entirety). In a typical thermal processing treatment, 1.4 mL ofAu_(2nm)-DT and Fe₂O₃ nanoparticles in toluene with various ratio wasplaced in a reaction tube. The mixed precursor solution containedAu_(2nm)-DT, OA- and/or OAM-Fe₂O₃ nanoparticles, toluene, and TOA-Br.The tube was then placed in a preheated Yamato DX400 Gravity ConvectionOven at 149° C. for 1-hour. Temperature variation from this set pointwas limited to ±1.5° C. After the 1-hour thermal treatment, the reactiontube was allowed to cool down and the particles were redispersed intoluene. The above approach can also be used to produce Fe₃O₄@Aunanoparticles. The term “Fe-oxide” was used to refer to a variety ofiron oxides, including Fe₂O₃ and Fe₃O₄.

Example 3 Preparation of Nanoparticles Capped with Proteins and SERSLabels

The as-synthesized DT-capped iron oxide@Au particles were transferred towater by ligand exchange using mercaptoundecanoic acid (MUA) byfollowing a procedure reported by Gittins et al. (Chem. Phys. Chem.3(1):110-113 (2002), which is hereby incorporated by reference in itsentirety), with a slight modification. The nanoparticles were furthermodified with DSP for protein coupling by ligand exchange. To 1 mLsolution of DT-capped iron oxide@Au particles (6 ng/mL) in borate buffer(pH 8.3), 140 μL of 1 mM DSP was added and stirred overnight. Thenanoparticles were rinsed with centrifuge and 20 μL of anti-Rabbit IgG(2.4 mg/mL) was pipetted. After overnight incubation, the particles werecentrifuged three times and finally resuspended in 2 mM Tris buffer (pH7.2) with 1% BSA and 0.1% Tween 80. The same method was also used forcoating protein A and BSA to Au nanoparticles of 80 nm size. 2.5 μL of 1mM DSP was added to 1 mL of Au particles (80 nm, 1×10¹⁰/mL) and reactedovernight. 40 μL of 50 mM borate buffer was added and either protein Aor BSA was added to make final concentration of protein A or BSA to be˜25 μg/mL. MBA was used as a Raman label (Ni et al., Anal. Chem.,71:4903 (1999), which is hereby incorporated by reference in itsentirety). To introduce spectroscopic label onto the Au nanoparticlesmodified with either protein A or BSA, an ethanolic solution of MBA (10μg/mL) was added and reacted overnight. Finally, the particles werecentrifuged three times and finally re-suspended in 2 mM Tris buffer (pH7.2) with 1% BSA and 0.1% Tween 80. The resulting protein cappednanoparticles were stored at 4° C.

Example 4 Measurements and Instrumentation

The study of the binding between Au nanoparticles labeled with protein A(or BSA) and MBA (A) and iron oxide@Au nanoparticles labeled withanti-rabbit IgG (B) was carried out by mixing (A) and (B). 250 μL of (A)was first diluted in 1750 μL tris buffer before mixing with ˜10 μL of(B). UV-vis spectra were collected immediately following gentle mixingof the solution. Spectroscopic measurements were performed after usingmagnet to collect the reaction product. For spectroscopic labeling, 0.8mL of ethanolic solution of MBA was added to 0.5 mL of iron oxide@Auparticles (30 mg/mL in toluene), and shaked overnight. The particleswere cleaned three times with toluene and ethanol and dispersed in 2 mMborate buffer. The particles were then drop cast on gold on micasurface.

Example 5 Surface-Enhanced Raman Scattering (SERS)

Raman spectra were recorded using the Advantage 200A Raman instrument(DeltaNu). The instrument collects data over 200 to 3400 cm⁻¹. The laserpower was 5 mW and the wavelength of the laser was 632.8 nm. In theexperiment, the spectrum in the range from 200 to 1500 cm⁻¹ wascollected.

Example 6 Transmission Electron Microscopy (TEM)

TEM micrographs of the particles were obtained using a Hitachi H-7000Electron Microscope operated at 100 kV. The particles dispersed inhexane were drop cast onto a carbon film coated copper grid followed byevaporation at room temperature.

Example 7 Ultraviolet-Visible Spectroscopy (UV-Vis)

UV-Vis spectra were acquired with a HP8453 spectrophotometer. Thespectra were collected over the range of 200-1100 nm.

Example 8 Direct Current Plasma-Atomic Emission Spectroscopy (DCP-AES)

The composition of synthesized particles and thin films was analyzedusing DCP-AES. Measurements were made on emission peaks at 267.59 nm and259.94 nm, for Au and Fe, respectively. The nanoparticle samples weredissolved in concentrated aqua regia, and then diluted to concentrationsin the range of 1 to 50 ppm for analysis. Calibration curves wereconstructed from standards with concentrations from 0 to 50 ppm in thesame acid matrix as the unknowns. Detection limits, based on threestandard deviations of the background intensity are 0.008 ppm and 0.005ppm for Au and Fe, respectively. Standards and unknowns were analyzed 10times each for 3 second counts. Instrument reproducibility, forconcentrations greater than 100 times the detection limit, results in<2% error.

Example 9 Morphological Characterization of the Core-Shell MagneticNanoparticles

Since the thermal processing treatment involved molecular desorption,nanoscrystal core coalescence, and molecular re-encapsulation processesat elevated temperatures, as demonstrated in previous work for gold andalloy nanoparticles (Schadt et al., Chem. Mater. 18:5147 (2006); Maye etal., Langmuir 16:490 (2000); Zhong et al., Chem. Commun. 13:1211 (1999),which are hereby incorporated by reference in their entirety), theexamination of the structure and morphology of the evolved nanoparticlesis important for the overall assessment of the viability in processingthe Fe-oxide@Au nanoparticles. As a control experiment, FIGS. 3A-B showthe representative set of TEM images for gold nanoparticles thermallyprocessed from 2-nm sized, DT-capped gold nanoparticles (2.0±0.4 nm).The increased particle size and the high monodispersity of the resultingnanoparticles (6.4±0.4 nm) are consistent with what has been previouslyreported, demonstrating the effectiveness of the thermal processingcondition for processing gold nanoparticles.

As another control experiment, the thermal processing of Fe₂O₃nanoparticles under the same condition was also examined. FIGS. 4A-Bshow a representative set of TEM images for Fe₂O₃ nanoparticles bythermally processing of Fe₂O₃ nanoparticles of 4.4 nm size. Theobservation of insignificant changes in the particle size and themonodispersity of the resulting nanoparticles (4.5±0.5 nm) demonstratethe absence of any size evolution for Fe₂O₃ nanoparticles with thethermal processing conditions used in the present work. The absence ofsize evolution is likely associated with the lack of significant changein melting temperature for Fe₂O₃ nanoparticles. In other words, Fe₂O₃nanoparticles are stable under the thermal processing temperature.

The above two control experiments demonstrate that the goldnanoparticles undergo thermally activated coalescence responsible forthe growth of Au nanoparticles whereas the Fe₂O₃ nanoparticles remainlargely unchanged in this process. In the next experiment, the thermalprocessing treatment of a controlled mixture of the 2-nm sized goldnanoparticles and the 4-nm sized Fe₂O₃ nanoparticles was examined underthe same thermal processing condition. Specifically, a toluene solutionof the two precursor nanoparticles (e.g., stock solutions ofdecanethiolate (DT)-capped Au (2 nm, 158 μM) and OAM and/or OA-cappedFe₂O₃ (5 nm, 6.3 μM), or Fe₃O₄ with a controlled ratio in a reactiontube was heated in an oven at 149° C. for 1 hour. Other constituents inthe solution included TOA-Br and DT with controlled concentrations.After cooling to room temperature, the solidified liquid was dispersablein toluene. FIGS. 5A-C show a representative set of TEM micrographscomparing the resulting nanoparticles obtained from the thermalprocessing treatment with the two precursor nanoparticles. It is evidentthat highly monodispersed nanoparticles with an average size of 6.2±0.3nm are obtained from the processing of the mixture using a 25:1 ratio ofAu to Fe₂O₃ nanoparticles (FIG. 5C). The precursor Fe-oxidenanoparticles were not observed after the processing treatment of themixture. This feature is in sharp contrast to the morphological featurescorresponding to the nanoparticle precursors of DT-capped Au (2.1±0.3nm) (FIG. 5B) and OA-capped Fe₂O₃ (4.4±0.3 nm) (FIG. 5A).

FIGS. 6A-B show another representative set of TEM images for thenanoparticles thermally evolved from the same precursors but with twodifferent molar ratios of the precursors, Au:Fe₂O₃=25:1 (FIG. 6A) and132:1 (FIG. 6B). It is evident that the size and monodispersity of theevolved nanoparticles are dependent on the precursor ratios. A higherratio of Au to Fe₂O₃ nanoparticle precursors clearly favors theformation of larger-sized and more monodispersed nanoparticles.

In addition to manipulating the relative concentrations of Au and Fe₂O₃to control the thickness of Au shell, the formation of the core-shellnanoparticles with multiple magnetic cores were shown to be possible,depending on the manipulation of a combination of control parameters.Nanoparticles with larger sizes (30-100 nm) have been obtained bycontrolling the ratio of the precursor nanoparticles and the chemicalnature of capping molecules. FIGS. 7A-D show a representative set of TEMimages for particles obtained with relatively-lower concentrations of Au(Au:Fe₂O₃=5:1), demonstrating that important roles have been played byboth the Au to Fe₂O₃ concentration ratio and the type of capping agenton the final size and monodispersity. It is also noted that the basicfeature of these evolved nanoparticles remains unchanged from sample tosample, demonstrating the reproducibility of the method. The averagesize for the nanoparticles obtained from OA-capped Fe₂O₃ is 31.4±3.0 nmwith high monodispersity (FIGS. 7A-B), whereas that from the OAM-cappedFe₂O₃ exhibits sizes of 30 to 100 nm and hexagon-shaped features (FIG.7C).

By controlling the condition so that the thermal equilibrium can beperturbed (e.g., shorter time, lower temperature, or larger reactionvolume), the observed features appear to correspond to the early stageof coalescence of the core-shell nanoparticles. For example, for thethermal evolution in a larger reaction volume, spherical clusters withhighly ordered packing morphology were observed (FIG. 7D). The center ofthe spherical assembly shows indications of interparticle coalescence,in contrast to the loosely-bound nanoparticles spread around thespherical outline. Control experiments showed that under the temperaturewhile Au nanoparticles could be evolved to sizes of up to 10 nm, Fe₂O₃nanoparticles remained unchanged. Thus, these large-sized particleslikely consist of multiple Fe-oxide cores.

Example 10 Characterization of the Surface Chemistry of the Core-ShellMagnetic Nanoparticles

There are two key questions that must be answered about the formation ofthe core-shell magnetic nanoparticles. The first question concernswhether surface of the nanoparticles are composed of Au shell, and thesecond question concerns whether the nanoparticle cores include magneticFe₂O₃ nanoparticles. To address these questions, both high-resolutionTEM (“HRTEM”) and electron diffraction (“ED”) techniques seemed to beideal for examining the detailed core-shell morphology. While smalldifferences in HRTEM and ED have been observed by comparing the Fe₂O₃@Aunanoparticles with the Au and Fe₂O₃ nanoparticles, the results were notconclusive. XPS technique is also not appropriate because of the depthsensitivity issue. It is also noted that XRD examination of thecore-shell nanoparticles was not conclusive either. It is possible thatthe lattice parameters of the iron oxide core were distorted by therelatively thick Au shell, which is a fundamental issue to be addressed.Detailed experimental data obtained from the examinations of thecore-shell nanoparticles is provided in terms of the surface chemistryof the Au shell and the magnetic properties of the core to address theabove two questions.

To prove that the resulting nanoparticles include the desired Au shell,two types of measurements were carried out, both of which were based onthe surface chemistry of gold-thiolate binding and core-shellcomposition. First, the core-shell composition was analyzed, for whichsamples were prepared by assembling the nanoparticles into thin films ona glass substrate using dithiols as linkers/mediators (FIG. 8) (Wang etal., J. Phys. Chem. B 109:21593 (2005); Leibowitz et al., Anal. Chem.71:5076 (1999); Luo et al., J. Phys. Chem. 108:9669 (2004), which arehereby incorporated by reference in their entirety). The dithiolate-goldbinding chemistry involves a sequence of exchanging, crosslinking, andprecipitation processes which has previously been demonstrated to occurto Au surface only. Thin film assemblies were observed for those with anAu surface, i.e., Fe₂O₃@Au (FIG. 8, (A)) and Au (FIG. 8, (B))nanoparticles (or a combination of (A) and (B)). In contrast, there wereno thin film assemblies for Fe₂O₃ nanoparticles under the same reactioncondition (FIG. 8, (C)).

To analyze the core-shell composition, samples of the dithiol-mediatedthin film assemblies of the nanoparticles were dissolved in aqua regia,and the composition was then analyzed using direct current plasma-atomicemission spectroscopy (DCP-AES) technique. The as-processednanoparticles were also analyzed for comparison. For example,1,9-nonanedithiol-mediated assembly of nanoparticles into a thin film(Brust et al., J. Chem. Soc. Chem. Commun. 1994:801-802, which is herebyincorporated by reference in its entirety) is selective to Au orFe₂O₃@Au but not to Fe₂O₃. As shown in Table 1, both Au and Fe weredetected, demonstrating that the nanoparticles contain both Fe and Aucomponents. It is therefore evident that the surface of Fe₂O₃ particlesmust be covered by Au.

TABLE 1 Analysis of Metal Composition in the Fe₂O₃@Au NanoparticlesAtomic ratio d_(shell) (nm) determined by Sample (Au:Fe) TEM DCP-AESAs-synthesized 77:23 1.1 1.0 Thin Film 84:16 1.1 1.4

Quantitatively, the Au:Fe ratios for the thin films were slightly higherthan those for the as-synthesized particles. The Au shell thickness canbe estimated from the Au:Fe ratios based on a spherical core-shell model(Wang et al., J. Phys. Chem. B 109:21593 (2005), which is herebyincorporated by reference in its entirety). The results obtained fromthe DCP data for both the as-synthesized and the thin film (Table 1) arefound to be very close to the values measured from the TEM data.

In the second type of measurements, SERS labels such as MBA wereimmobilized on the surface of the core-shell nanoparticles viaAu-thiolate binding chemistry. FIGS. 9A-B show a representative set ofSERS spectra for the core-shell Fe₂O₃@Au nanoparticles labeled with MBA.The SERS for Fe₂O₃@Au nanoparticles (FIG. 9A) showed clearly two peaksat 1084 and 1593 cm⁻¹, which are identical to those observed for the Aunanoparticles (FIG. 9B). These two bands correspond to ν(CC)ring-breathing modes of MBA, which are characteristic of the expectedsignature (Varsanyi, Assignments for Vibrational Spectra of SevenHundred Benzene Derivatives; John Wiley & Sons: New York, 1974, which ishereby incorporated by reference in its entirety). Controlled experimentwith Fe₂O₃ nanoparticles incubated with MBA did not show any of thesebands. This observation demonstrates that MBA labels are immobilized onthe Au shell of the Fe₂O₃@Au nanoparticles.

Example 11 Protein Binding and Bioseparation Using the Core-ShellMagnetic Nanoparticles

For the targeted bio-separation application, both the protein-bindingproperties of the gold shell and the magnetic properties of the core inthe Fe₂O₃@Au nanoparticles are essential. The recent results of magneticcharacterization for similar core-shell Fe-oxide@Au nanoparticles (Wanget al., J. Phys. Chem. B 109:21593 (2005), which is hereby incorporatedby reference in its entirety) have revealed detailed information forassessing the magnetic properties. In FIGS. 10A-C, a set of photos isshown to illustrate the movement of the nanoparticles dispersed insolutions before and after applying a magnetic field (NdFeB type) to theparticles. The core-shell nanoparticles can be fully dispersed intoluene solution (FIG. 10A), which did not respond to external magneticfield of the magnet. However, the suspension of the same nanoparticlesin ethanol-toluene (FIG. 10B), which has an increased magneticsusceptibility due to aggregation, showed that the suspended particlesmoved towards the wall near the magnet gradually (FIG. 10C), eventuallyleaving a clear solution behind.

This test result demonstrated clearly that the Fe₂O₃@Au nanoparticlesare magnetically active, which is desired for the targetedbio-separation application. Similar results were also obtained for thecore-shell nanoparticles prepared using different precursor ratios.

To further demonstrate the viability of the Fe₂O₃@Au nanoparticles formagnetic bio-separation, the following proof-of-concept demonstrationexperiment exploits both the magnetic core and the bio-affinity of thegold shell. In this experiment, the gold-based surface protein-bindingreactivity and the Fe-oxide based magnetic separation capability wereexamined. The DT-capped Fe₂O₃@Au particles were first converted towater-soluble particles by ligand exchange reaction with MUA. The ligandexchange reaction involved replacement of the original capping molecules(OAM and OA) on Fe₂O₃@Au nanoparticles by acid functionalized thiols. Toprove the ligand exchange on the nanoparticle surface, FIGS. 11A-B showa representative set of FTIR spectra of the OAM/OA-capped Fe₂O₃@Aunanoparticles before and after the ligand exchange reaction. After theexchange reaction, the band at 3004 cm⁻¹ corresponding to the C—Hstretching mode next to the double bond from OA and OM capping molecules(FIG. 11A) are clearly eliminated (FIG. 11B). In the meantime, the bandat 1709 cm⁻¹ corresponding to the carboxylic acid group of MUA isdetectable after the exchange reaction. Furthermore, the spectral changein the 1300-1560 cm⁻¹ region seemed to support the presence of bandscorresponding to the symmetric and asymmetric stretching modes in thecarboxylate groups of MUA.

These observations demonstrate the successful exchange of the MUA withthe original ligands on the nanoparticles. This is further evidenced bythe fact that the post-exchanged core-shell nanoparticles becamewater-soluble.

The MUA-capped Fe₂O₃@Au nanoparticles in water underwent furtherexchange reaction with a protein-coupling agent, DSP, forming aDSP-derived monolayer on the gold surface. Antibody (anti-rabbit IgG)was then immobilized onto the resulting nanoparticles via coupling withthe surface DSP, forming Ab-immobilized core-shell nanoparticles.

The Ab-immobilized core-shell nanoparticles were reacted with Auparticles capped with both protein-A and a Raman label, e.g., MBA. FIG.12 illustrates the reactions and product separation of theantibody-labeled Fe₂O₃@Au nanoparticles in two different reactionsystems: reaction with protein A capped gold nanoparticles (A1-B1), andreaction with BSA capped gold nanoparticles (A2-B2). In each case,magnetic field was applied to collect the magnetically-active productsfor SERS analysis.

FIG. 13 shows a representative set of UV-Vis spectra monitoring thereaction progress. Results from control experiments are also includedfor comparison, in which the Au particles capped with BSA (bovine serumalbumin) and MBA were used to replace the Au particles capped withprotein A while maintaining the rest of the conditions. The specificreactivity between Fe₂O₃@Au nanoparticles capped with anti-rabbit IgG(Ab) and Au nanoparticles capped with protein-A is clearly evidenced bythe gradual decrease of the surface plasmon (SP) resonance band at 535nm and its expansion at the longer wavelength region (A1). This findingis in sharp contrast to the lack of any change in SP band for thereaction between Fe₂O₃@Au nanoparticles capped with anti-rabbit IgG andAu nanoparticles capped with BSA (A2). In contrast to the specificbinding of anti-rabbit IgG to protein-A, there is no specific bindingbetween BSA and anti-rabbit IgG.

FIG. 14 shows a representative set of SERS spectra of the productscollected by applying the magnet to the reaction solution for theFe₂O₃@Au nanoparticles capped with anti-rabbit IgG and Au nanoparticlescapped with protein-A. The spectrum from the control experiment is alsoincluded for comparison, in which the Au nanoparticles capped with BSAprotein and MBA label were used to react with Fe₂O₃@Au nanoparticlescapped with anti-rabbit IgG while maintaining the reaction conditions.

The product of the reaction collected by a magnet showed the expectedSERS signature of MBA (B1), corresponding to ν(C—C) ring-breathing modesof MBA. This finding is in sharp contrast to the absence of SERSsignature in the control experiment (B2). These two findings clearlydemonstrate the viability of exploiting the magnetic core and gold shellof the Fe₂O₃@Au nanoparticles for interfacial bio-assay and magneticbio-separation.

In conclusion, a novel strategy based on the thermally-activatedhetero-coalescence between Au and Fe₂O₃ nanoparticles has beendemonstrated for the fabrication of monodispersed magnetic core-shellnanoparticles, Fe₂O₃@Au. Similar results have also been observed forFe₃O₄ nanoparticle cores. In comparison with the previous sequentialsynthesis method involving reduction and deposition of gold ontopre-synthesized iron oxide nanoparticles (Wang et al., J. Phys. Chem. B109:21593 (2005), which is hereby incorporated by reference in itsentirety), the nanoparticles made by the thermal approach can havesimilar core-shell structure with gold shell and iron-oxide core, andshow similar behavior in forming thin films due to the gold shell. Someof the major differences between the two nanoparticle products includethe core-shell size range and the surface capping structure. Due to theunique processing environment, nanoparticles with much larger sizes canbe made. The capping structure can be tailored differently in the twocases. These core@shell nanoparticles consist of magnetically-activeFe-oxide core and thiolate-active Au shell, which were shown to exhibitthe Au surface binding properties for interfacial biological reactivityand the Fe-oxide core magnetism for magnetic bio-separation. Thesemagnetic core-shell nanoparticles have therefore shown the viability forutilizing both the magnetic core and gold shell properties forinterfacial bio-assay and magnetic bio-separation. These findings areentirely new, and could form the basis of fabricating size, magnetism,and surface tunable magnetic nanoparticles for bio-separation andbiosensing applications.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed:
 1. Core-shell nanoparticles, each core-shellnanoparticle comprising: a silver shell; a plurality of discrete,nonconcentric, iron oxide-containing cores, each core being within andsurrounded by said silver shell; and a ligand cap over said silvershell.
 2. The core-shell nanoparticles according to claim 1, wherein thenanoparticles are present in a monodispersion with controlled diametersranging from 5 nm to 100 nm.
 3. The core-shell nanoparticles accordingto claim 1, wherein the iron oxide-containing cores are magnetic,paramagnetic, or superparamagnetic.
 4. The core-shell nanoparticlesaccording to claim 1 wherein the iron oxide-containing core comprises acompound selected from the group consisting of Fe₃O₄ and Fe₂O₃.
 5. Thecore-shell nanoparticles according to claim 4, wherein the ironoxide-containing core comprises Fe₂O₃.
 6. The core-shell nanoparticlesaccording to claim 4, wherein the iron oxide-containing core comprisesFe₃O₄.
 7. The core-shell nanoparticles according to claim 1, wherein theligand cap is selected from the group consisting of decanethiolate,oleylamine, oleic acid, acrylates,N,N-trimethyl(undecylmercapto)ammonium (TUA), tetrabutylammoniumtetrafluoroborate (TBA), tetramethylammonium bromide (TMA),cetyltrimethylammonium bromide (CTAB), citrates, poly methacrylate,ascorbic acid, DNA, 2-mercaptopropionic acid (MPA), 3-mercaptopropionicacid (MPA), 11-mercaptoundecanoic acid (MUA),10-mercaptodecane-1-sulfonic acid, 16-mercaptohexadecanoicacid, diimide,N-(2-mercaptopropionyl)glycine (tiopronin), 2-mercaptoethanol,4-mercapto-1-butanol, dodecyl sulfate, amino acids, homocysteine,homocystine, cysteine, cystine, glutathione, mercaptobenzoic acid (MBA),Protein A, bovine serum albumin (BSA), and anti-rabbit-IgG (Ab).
 8. Thecore-shell nanoparticles according to claim 7, wherein the ligand cap isselected from the group consisting of decanethiolate, oleylamine, andoleic acid.
 9. The core-shell nanoparticles according to claim 8,wherein the ligand cap is decanethiolate.
 10. The core-shellnanoparticles according to claim 8, wherein the ligand cap isoleylamine.
 11. The core-shell nanoparticles according to claim 8wherein the ligand cap is oleic acid.
 12. The core-shell nanoparticlesaccording to claim 1, wherein the ligand cap is tetraoctylammoniumbromide.
 13. The core-shell nanoparticles according to claim 1 furthercomprising: a first binding material bound to the ligand-capped shell.14. The core-shell nanoparticles according to claim 13, wherein thefirst binding material is selected from the group consisting ofproteins, peptides, antibodies, and antigens.
 15. The core-shellnanoparticles according to claim 1, wherein the iron oxide-containingcore comprises Fe₃O₄ and the ligand cap is tetraoctylammonium bromide.